Opioid-induced glial activation, which compromises pain treatment and contributes to the development of drug addiction and abuse, is regulated via a signaling pathway downstream of toll-like receptor-4 (TLR4), a membrane spanning receptor that functions in complex with its accessory protein MD-2. As current opioid pharmacotherapeutics have failed to control pain while avoiding the negative consequences, there is an urgent need to understand opioid dysregulation via TLR4. The central hypothesis of the current proposal is that disruption of the TLR-4/MD-2 complex formation can inhibit opioid-induced glial activation, thereby enhancing analgesia and reducing opioid tolerance and dependence. The rationale underlying the proposed research is that the identified TLR4 inhibitors, which selectively block the critical protein-protein interactions between TLR4 and MD-2, will provide a useful tool for investigating the role of the TLR4-mediated signaling pathway in glial activation. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation. The proposed high risk/high reward approach, if successful, is projected to yield significant novel outcomes. First, the results will shed light on the mechanism of the clinically relevant opioid-induced glial activation. Second, if successful, the peptide and peptidomimetic antagonists of the TLR4/MD-2 interactions identified in the proposed research can serve as prototypes for more drug-like small-molecule inhibitors. These inhibitors may eventually find application in the development of novel therapeutics to enhance the clinical efficacy of opioid analgesics and to treat opioid addiction and abuse, as well as other clinically relevant indications. The proposed studies are built on the complementary strength of the PI in the design, synthesis, and evaluation of novel protein-protein interaction inhibitors, and of the Co-PI, who has extensive expertise in glial neurobiology and will provide support in animal testing of the identified inhibitors. In Aim 1, antagonists of TLR4 that block the TLR4/MD-2 complex formation will be developed. The working hypothesis here is that conformationally strained peptides derived from the TLR4-binding region of MD-2 can compete with the full-length MD-2 protein and thereby inhibit the TLR4/MD-2 interaction. In Aim 2, the second working hypothesis, that the inhibitors of the TLR4/MD-2 interactions can non-competitively antagonize opioids to block TLR4-mediated glial activation, will be tested. Cellular assays and animal models will be used to evaluate the inhibition of glial activation by the TLR4 antagonists both in vitro and in vivo. The proposed research is significant because it is expected to establish the TLR4/MD-2 protein-protein complex as a novel therapeutic target for preventing and treating opioid abuse. Regarding its positive impact on scientific advancements, this work will (1) improve scientific understanding of drug dependence and pain suppression and (2) allow the development of a new generation of therapeutics. PUBLIC HEALTH RELEVANCE: We aim to unravel the mechanism of opioid-induced glial activation that importantly contributes to the development of drug addiction and abuse. As an outcome of the proposed investigations, we expect to attain a better understanding of the molecular mechanisms of opioid-induced glial activation, and to clarify novel targets to regulate such activation. The intellectual merit of the proposed work lies not only in its scientific advancements in the field of chemical biology and protein engineering, but also in its potential impact on clinical applications. This work will (1) improve scientific understanding of drug dependence and pain suppression;and (2) allow the development of a new generation of therapeutics.